This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cullin RING ligases (CRLs) comprise the largest subfamily of E3 ubiquitin ligases. In humans, six cullins (CUL1, 2, 3, 4A, 4B, and 5), two RBX-family RING proteins (RBX1 and 2), and hundreds of substrate receptors assemble into distinct CRLs that mediate ubiquitination of thousands of targets to regulate a vast array of cellular processes. CRL function is regulated by attachment of the ubiquitin-like protein (UBL) NEDD8 to a conserved Lys in a cullin's C-terminal domain. NEDD8 both enhances intrinsic CRL ubiquitination activity, and prevents CRL binding to the inhibitor CAND1. At present, the NEDD8 cascade is known to contain a single E1 (NAE1-UBA3), which activates NEDD8 and ultimately catalyzes transfer of NEDD8's C-terminus to the catalytic cysteine of the characterized NEDD8 E2, UBE2M (also known as UBC12). The resulting UBE2M~NEDD8 thioester conjugate serves as the direct source of NEDD8 to be covalently attached to a cullin's acceptor Lys. Despite the importance of CRL activation by NEDD8, it remains unknown whether there is any specificity in cullin NEDD8ylation, or how such specificity could be established. Clues to selectivity might come from analogy to the ubiquitin (Ub) pathway. In vertebrates, the Ub pathway comprises two E1s, tens of E2s and hundreds of E3s promoting Ub transfer to thousands of targets. Within this hierarchy, E1-E2 and E2-E3-substrate interactions are highly specific to ensure precise temporal and spatial regulation of targets by modification with appropriate Ub linkages, which in turn mediate particular downstream functions. In contrast to the Ub pathway, the NEDD8 conjugation cascade was been reported to contain only a single E2. Given the important and diverse CRL functions in cellular regulation, our goal is to understand whether, and if so how, the NEDD8 cascade is equipped with additional levels of selectivity for cullin NEDD8ylation.